Our studies focus of the Escherichia coli mechanism of chemotaxis to oxygen (aerotaxis) and related responses, such as redox taxis and glycerol taxis. The responses are different from other chemotactic behaviors in requiring a functional electron transport system. Aer, a novel flavoprotein, and Tsr, the serine chemoreceptor, have been recently identified by this laboratory as the transducers for oxygen, redox and glycerol (energy) taxis. The long term goal is to understand, in molecular detail, the signal processing by thee transducers. The specific aims include: 1) Test the hypothesis that flavin adenine dinucleotide (FAD) in Aer senses redox changes Date Released:05/07/1997 Date Printed: 10/22/1997 in the electron transport system, and Tsr senses proton motive force. 2) Investigate the relationship between structure and function of Aer and Tsr in sensing redox potential and proton motive force. 3) Test the hypothesis that proton motive force/redox potential is a signal that is common to aerophobic and aerophilic responses in E. Coli, guiding bacteria to an optimal environment where the proton motive force is maximal. An interdisciplinary approach - combining contemporary methods of molecular biology, genetics and biochemistry with novel techniques developed in this laboratory - will be used to address the critical research questions identified for each aim. The electron transport system will be perturbed in E. Coli using newly available constructs. Putative residues for FAD binding in Aer will be mutated by site directed mutagenesis and the effect on aerotaxis, FAD binding and mid-point reduction potential determined. The topology of Aer in the membrane will be determined using a sandwich Aer fusion protein in which PhoA or LacZ is inserted in frame within the N-terminal domain of Aer. Cysteine scanning of H328, selected histidine residues in the periplasmic domain and residues around the K1 coiled coil domain of Tsr will be used to identify the Ph receptors and their effect on proton motive force (redox) sensing by Tsr. The hypothesis that aerotaxis guides bacteria to the oxygen concentration that supports the highest proton motive force will be tested using simultaneous measurement of oxygen concentration and membrane potential, under conditions where the oxygen concentration can be tightly controlled. Elucidating these mechanisms will provide important insights into a primordial pathway for chemotaxis, and also into general principles of oxygen sensing (some of which may be applicable to carotid body chemoreceptors in humans).